| Home | E-Submission | Sitemap | Contact Us |  
Environ Eng Res > Volume 26(3); 2021 > Article
Bae: Effects of different cell states of Microcystis aeruginosa on coagulation process


Three different states of Microcystis aeruginosa, untreated cells, pure cells, and broken cells, were tested for coagulation efficiency to understand their effects on drinking water treatment facilities. Microcystis aeruginosa in its late log-growth phase to early stationary phase, day 12 of culture in the M11 medium, were collected since cells are the most active and reach its maximum population during those periods. Untreated cells samples showed worst results for turbidity removal while turbidities for pure cells and broken cells ones presented the similar levels with those for 0 cell samples after 3 Al-mg/L of coagulant dose. Organic matters from broken cells were not properly removed throughout coagulation processes since samples with organic matters - untreated cells and broken cells - showed higher levels of E254 and E260 after jar-tests than pure cell samples did. Overall, coagulation processes were highly interrupted when the Microcystis aeruginosa cells and organic matters from cells were co-existed. In addition, algae originated matters which may not remove by coagulation process could lead the secondary pollution when algae are introduced into drinking water treatment facilities during waterbloom. Therefore, the facility should pay attention on the results since they have to assure their water quality for public health.

1. Introduction

Urbanization and population growth lead increasing input of sewage and wastewater to the waterbodies and result in decreasing water quality, water pollution. Water pollution occurs when contaminants exceeding the capacity of the self-purification of water system and one of typical water pollution is eutrophication. The term “eutropic” is derived from the Greek word “rich in nutrients” and refers to the deterioration of water quality caused by excessive inorganic nutrients and/or organic matters in the waterbodies.
Phytoplankton and algae will dramatically increase once waterbodies are eutrophicated, Especially, cyanobacteria (blue-green algae) will be the dominant strain when temperature is high and this is so called the waterbloom. During waterblooms, cyanobacteria will cover the most of water surfaces and thihs causes blocking oxygen transfer between air and waterbody, perishing aquatic organism as well as generating unpleasant odor and taste. Some cyanobacteria, moreover, produce toxic substances such as hepatotoxin, nevertoxin, and cytotoxin which are causes of dermatitis, stomachache, headache and allergy of human being as well as the death of aquatic organism and mammals [15]. Furthermore, waterblooms by cyanobacteria are the cause of interrupting water treatment process, i.e. inhibiting coagulation process, shortening filter’s longevity, increasing pH level, decreasing dissolved oxygen level, etc. [68].
Microcystis aeruginosa is the most common strain while waterblooms occur not only in Korea, but also in worldwide. In fact, toxic damages caused by Microcystis aeruginosa during waterblooms have been reported worldwide [912]. Microcystis aeruginosa produces microcystin, one of hepatotoxin, and the toxin may cause severe injury to the liver since it is already reported that hepatotoxins are the cause of the clinical consequences for acute or chronic liver injury, with the possibility of enhanced susceptibility to, and growth of, liver tumors [13]. Furthermore, Microcystis aeruginosa inhibit the water treatment process, especially for the coagulation process and filtration process [1416]. Inappropriate managements, therefore, for Microcystis aeruginosa during drinking water treatment process may bring serious public health problems.
In this study, coagulation efficiencies for different algae cell status was investigated to understand whether coagulation inhibitions would be caused by algae cell itself or by organic matters originated from algae. The results would provide better ideas to water treatment facilities when waterbloom occurs in their water resources.

2. Material and Methods

2.1. Cultivation of Microcystis Aeruginosa

Microcystis aeruginosa was obtained from Korean National Institute of Environmental Research and cultured in M11 medium, most widely used for culturing Microcystis aeruginosa [1720]. Table 1 shows the composition of M11 medium. Microcystis aeruginosa were collected from its late log-growth phase to early stationary phase, specifically, day 12 of culture when algae reached their maximum cell numbers, approximately 3.4 × 108 cell/mL, since algae in this period should be the most active and release the most abundant organic matters.
Followings are the cell counting process for the study; 0.1 mL of 12% NaClO was added in 100 mL of cultured water as well as coagulated water and left them one hour at room temperature as the pre-treatment. Water samples were sonicated with 30 W for 5 min to make cells fall apart after pre-treatment and then Hemocytometer, broadly used for microcell counting, were used to count cell numbers of Microcystis aeruginosa [21, 22].

2.2. Preparing Different Cell States

Three different states of algae cells were prepared for the study; 1. untreated cells 2. pure cells 3. broken cells. Cells in M11 medium were used as they were for untreated cell samples. Untreated cells contain pure cells and External Organic Matters (EOMs). Pure cells were obtained by centrifuging (4,000 g, 15 min) fully grown cells in their late log-growth phase to early stationary phase at M11 medium. The centrifugation was repeated one more time after the supernatant from the first centrifugation was discarded and then samples were filled with distilled water. Broken cells were getting from destroying cell membrane by using high frequency sounds (20 kHz, 100 W, 10 min) and then samples were checked whether or not there might be unbroken cells with microscopic observation. The process was kept repeating until all cells were totally broken. Broken cell samples contain Internal Organic Matters(IOMs), Surface Organic Matters (SOMs), and EOMs [2].

2.3. Jar-Test

500 mL of water samples were used for jar-test. Distilled water was used to rule out the effects of other factors except algae cell itself. Turbidity and alkalinity of water samples were artificially adjusted using Kaolin and CaCO3; Turbidity 40 NTU and Alkalinity 100 mg/L of CaCO3. Each state of Microcystis aeruginosa were added in water samples after turbidity and alkalinity were adjusted. Jar-tests were carried out with 140 rpm for first 10 min and 40 rpm for following 15 min and then water samples were left for 30 min to settle down flocks. Jar-tests were repeated with every 1 Al-mg increment of coagulant dose, 1 Al-mg to 18 Al-mg, for each state of cell sample and control one. Al2(SO4)2·16~18H2O (Alum) were used as coagulant and pH levels of tested water were adjusted to stay their levels at 7 ± 0.05 using 1 N and 0.01 N of NaOH and HCl during jar-tests.

2.4. Evaluation for Efficiency of Coagulation Process

Turbidity, UV254, UV260 and residual aluminum were analyzed after each jar-test to evaluate efficiencies of coagulation process for removal of each cell state of Microcystis aeruginosa based on Drinking Water Quality Standard Methods [23]. Shimadzu UV spectrophotometer (UV 1800 model) was used to analyze turbidity, UV254, and UV260. Shimadzu atomic absorption spectrophotometer (AA 6300 model) was used to determine concentrations of residual aluminum in water samples and coagulated water.

3. Results and Discussion

Several factors were analyzed after finishing jar-tests to find coagulation efficiencies for each state of algae cell and followings are the results.

3.1. Turbidity in Jar-Tested Water

Fig. 1 shows jar-test results for turbidity changes of each cell state sample and control dependent on coagulant dose. Control (line with squares) represents 0 cell/L distilled water samples adjusted turbidity and alkalinity.
For controls, a rapid turbidity removal was observed with the first jar-test, 1 Al-mg coagulant dose and the additional removal was shown with the right next jar-test, 2 Al-mg coagulant dose. Turbidity removals for other jar-tests, over 3 Al-mg coagulant dose, were slightly better than that for the second jar-test, 2 Al-mg coagulant dose and stayed in similar levels. Patterns of turbidity removal for broken cell samples were similar to those for control. The results indicate that coagulation process may not inhibit if algae cells are not existed. Jar-tests over 3 Al-mg coagulant dose for pure cell samples had similar turbidity removal rates compared to control and broken cell samples, but jar-tests with 1 and 2 Al-mg coagulant dose for pure cell samples showed worse turbidity removal rates. Pure cells, therefore, may inhibit coagulation processes more than organic matters from Microcystis aeruginosa do.
Lastly, turbidities for coagulated water of untreated cell samples showed higher levels than those of other samples although coagulant doses were increased which results showed that coagulation inhibitions were greater when cells and organic matters from Microcystis aeruginosa coexist rather than cells or organic matters are present singly. The similar result could be found in the previous study which reported that co-existence of algae cells and algae originated matter (AOM) may cause of decreasing coagulation efficiency; 95% of algae cells can be removed by coagulation process while AOMs may not be removed [24]. Treating, therefore, either algae cells or organic matters released from algae might significantly reduce coagulation inhibitions when dealing with water resources during waterbloom.

3.2. Residual Aluminum in Jar-Tested Water

To check the absorption capability of coagulant towards Microcystis aeruginosa cell itself or organic matters, residual aluminum were measured after jar-tests and Fig. 2 shows concentrations of residual aluminum in coagulated water for all samples.
Residual aluminum concentrations of coagulated water for control samples showed the lowest levels and those for pure cell samples did the highest as the previous study showed that the increase in algae count and turbidity causes the rise of the concentrations of residual alumimum [25]. The results are considered to be due to the fact that algae cell sizes are much bigger than those of AOMs or kaolin particles. In other words, surface areas of pure cells which could contact coagulants are smaller than those of organic matters or kaolin particles, so that pure cell samples require less coagulants than other samples. Additionally, kaolin particles agglomerate around large algae cells to form larger flocs to settle down which also makes less coagulant consumptions.
Another issue is that residual aluminum concentrations for jar-tests over 3 Al-mg coagulant dose remained at similar levels and same patterns were shown for turbidity reductions. The 3 Al-mg/500 mL of coagulant, therefore, is the optimum dose for turbidity removal and residual aluminum.

3.3. E254 and E260 of Jar-Tested Water

E254 and E260 indicate UV254 and UV260 related substance, respectively. Specific UV wavelengths, UV254 and UV260, are used to determine the amount of organic matter in water. E254 related to UV254 indicates aromatic compounds is an indirect indicator for Trihalomethans (THMs, one of carcinogens) precursors and E260 related to UV260 indicates for biodegradable organic matters is an indirect indicator for persistent organic pollutants (POPs) in the water [2628].
The study focuses on E254 and E260 since byproducts of Microcystis aeruginosa have been known to contain these organic matters [2]. Algae cells release the most abundant E254 and E260 during their log-growth phase and stationary phase rather than their lag phase and death phase and this is another reason why the study focused on cells in log-growth phase and stationary phase since they may affect most on drinking water treatment systems [29].
As mentioned above, AOMs may not be removed although algae cell itself could be eliminated by coagulation process [24]. Pre-treatment such as photocatalytic before coagulation process could increase the removal efficiency for AOMs [30].
Fig. 3 and Fig. 4 show changes of E254 and E260 in jar-tested water. Changes of E254 and E260 in jar-tested water for all samples had comparable patterns to each other. It is obvious that samples with organic matters - untreated cells and broken cells - showed higher levels of E254 and E260 after jar-tests than pure cell samples did. Levels of E254 and E260 for pure cell samples were even lower than those for controls. As results, SOMs may not related to E254 and E260 because pure cell samples only have SOMs while others have IOMs and/or EOMs including SOMs. In addition, E254 and E260, precursors of THMs and POPs, are more in EOMs than IOMs since both untreated cells and broken cells have EOMs.

4. Conclusions

The study examined coagulation characteristics for different cell states of Microcystis aeruginosa, most common species during water blooms, to give better ideas for researchers, managers and operators who focused on drinking water treatment facilities when waterbloom occurs on their water resources. Coagulation characteristics of Microcystis aeruginosa revealed different behaviors in each cell state.
For the single existence of pure cells or organic matters, broken cell samples showed better turbidity removal rates than pure cell samples did, so pure cells may inhibit coagulation processes more than organic matters from Microcystis aeruginosa do. Turbidities for coagulated water of untreated cell samples showed higher levels than those of other samples in all jar-tests with different coagulant doses which results showed that coagulation inhibitions were greater when cells and organic matters coexist rather than cells or organic matters are present individually. Treating, therefore, only one of algae cells or organic matters released from algae might significantly reduce coagulation inhibitions when waterbloom occurs in water resources.
Pure cell samples showed the highest concentrations for residual aluminum after jar-test. Facts that algae cells are bigger than algae-derived organisms or kaolin particles and kaolin particles agglomerate around large algae cells to form larger flocs to settle down makes less coagulant consumptions.
Also, residual aluminum concentrations after jar-tests were inversely proportional to turbidity removal rates, but their patterns were analogous; their concentrations remained in similar levels for jar-tests over 3 Al-mg coagulant dose. The optimum coagulant dose, therefore, for turbidity removal is 3 Al-mg/500mL, so the coagulant dose should exceed its critical point to obtain better removal rates, but should not exceed it by too much for the sake of cost efficiency.
The removal patterns for E254 and E260 of all cell states samples were similar. Samples with organic matters showed higher E254 and E260 and levels of E254 and E260 for pure cell samples were even lower than those for controls. SOMs, therefore, may not related to E254 and E260 and EOMs may be the main sources of E254 and E260 since both untreated cells and broken cells have EOMs. The results indicate that water treatment facilities should pay special attentions to EOMs, so that they could remove potential risks for public health from Microcystis aeruginosa and its byproducts. Moreover, it is better to remove organic matters rather than algae cell itself in order to reduce the coagulation inhibition since organic matters could have potential risks to the public health.
The study will assist water treatment facilities in developing specific techniques or processes during waterbloom to reduce potential risks for public health as well as water treatment process inhibitions from cyanobacteria and its byproducts.


Author Contributions

H.K.B. (Professor) conducted all experiments and analyzed all data and results.


1. Liu Q, Zhang G, Ding J, Zou H, Shi H, Huang C. Evaluation of the Removal of Potassium Cyanide and its Toxicity in Green Algae (Chlorella vulgaris). Bull Environ Contam Toxicol. 2018;100:228–233.
crossref pdf

2. Jin JS. The effective removal of Microcystis aeruginosa and its by-products in drinking water treatment plants [dissertation]. Daegu: Keimyung University; 2002.

3. Turner PC, Gammie AJ, Hollinrake K, Codd GA. Pneumonia associated with contact with cyanobacteria. BMJ. 1990;300:1440–1441.

4. Long EG, Ebrahimzadeh A, White EH. Alga associated with diarrhea in patients with acquired immunodeficience syndrome and travelers. J Clin Microbiol. 1990;28:1101–1104.

5. Keleti G, Sykora JL, Maiolie LA, Doerfler DL, Campbell IM. Isolation and characterization of endotoxin from cyanobacteria (blue-green algae). The Water Environment: Algal Toxins and Health. Carmichael WW, editor1st edSpringer; 1981. p. 447–464.

6. Buratti FM, Manganelli M, Vichi S. , et alCyanotoxins: producing organisms, occurrence, toxicity, mechanism of action and human health toxicological risk evaluation. Arch Toxicol. 2017;91:1049–1130.
crossref pdf

7. Takaara T, Sano D, Konno H, Omura T. Cellular proteins of Microcystis aeruginosa inhibiting coagulation with polyaluminum chloride. Water Res. 2007;41:1653–1658.

8. Daly RI, Ho L, Brookers JD. Effect of chlorination on Microcystis aeruginosa cell integrity and subsequent microcystin release and degradation. Environ Sci Technol. 2007;41:4447–4453.

9. Yeom HS, Son H, Ryu JS, Jung EY, Kim KA. Release of Microcystin from Microcystis sp. by Pre-Oxidation and Removal of Microcystin by Advanced Drinking Water Treatment. J Korean Soc Environ Eng. 2019;41:532–540.
crossref pdf

10. Kim IS, Nguyen GH, Kim S, Lee J, Yu HW. Evaluation of Methods for Cyanobacterial Cell Lysis and Toxin (Microcystin-LR) Extraction Using Chromatographic and Mass Spectrometric Analyses. Environ Eng Res. 2009;14:250–254.
crossref pdf

11. Luukkainen R, Sivonen K, Namikoshi M, Färdig M, Rinehart KL, Niemelä SI. Isolation and identification of eight microcystin from thirteen Oscillatoria agardhii strains and structure of a new microcystin. Appl Environ Microbiol. 1993;59:2204–2209.

12. Sivonen K, Namikoshi M, Evans WR. , et alIsolation and characterization of a variety of microcystins from seven strains of the cyanobacterial genus Anabaena. Appl Environ Microbiol. 1992;58:2495–2500.

13. Falconer IR, Burch MD, Steffensen DA, Choice M, Coverdale OR. Toxicity of the blue-green alga (cyanobacterium) Microcystis aeruginosa in drinking water to growing pigs, as an animal model for human injury and risk assessment. Environ Toxicol. 1994;9:131–139.

14. Yang B, Park JA, Nam HL. , et alDegradation of Microcystin-LR, Taste and Odor, and Natural Organic Matter by UV-LED Based Advanced Oxidation Processes in Synthetic and Natural Water Source. J Korean Soc Environ Eng. 2017;39:246–254.
crossref pdf

15. Li S, Tan H-Y, Wang N. , et alThe Role of Oxidative Stress and Antioxidants in Liver Diseases. Int J Mol Sci. 2015;16:26087–26124.

16. Cadel-Six S, Moyenga D, Magny S, Trotereau S, Edery M, Krys S. Detection of free and covalently bound microcystins in different tissues (liver, intestines, gills, and muscles) of rainbow trout (Oncorhynchus mykiss) by liquid chromatography-tandem mass spectrometry: Method characterization. Environ Pollut. 2014;185:333–339.

17. Chu Z, Jin X, Iwami N, Inamori Y. The effect of temperature on growth characteristics and competitions of Microcystis aeruginosa and Oscillatoria mougeotii in a shallow, eutrophic lake similar system. Qin B, Liu Z, Havens K, editorsEutrophication of Shallow Lakes with Special Reference to Lake Taihu, China. Developments in Hydrobiology. 194:Dordrecht: Springer; 2007. p. 217–223.

18. Ueki M, Matsui K, Choi K, Kawabata Z. The enhancement of conjugal plasmid pBHR1 transfer between bacteria in the presence of extracellular metabolic products produced by Microcystis aeruginosa. FEMS Microbiol Ecol. 2004;51:1–8.
crossref pdf

19. Imai A, Fukushima T, Matsushige K. Effects of iron limitation and aquatic humic substances on the growth of Microsystis aeruginosa . Can J Fish Aquat Sci. 1999;56:1929–1937.

20. Fujimoto N, Sudo R, Sugiura N, Inamori Y. Nutrient-limited growth of Microcystis aeruginosa and Phormidium tenue and competition under various N:P supply ratios and temperatures. Limnol Oceanorg. 1997;42:250–256.

21. Khoddami M, Farzaneh M, Anaraki MG. Diagnosis of urinary tract infection using standard urinalysis or Hemocytometer leukocyte count. Iran J Pathol. 2006;1:117–120.

22. Huffman PA, Arkoosh MR, Casillas E. Characteristics of peripheral blood cells from rainbow trout evaluated by particle counter, image analysis, and Hemocytometric techniques. J Aquat Anim Health. 1997;9:239–248.

23. KME (Korea Ministry of Environment). Drinking water quality standard methods. Announcement of Ministry of Environment; Korea: Announcement No. 2009-2332010.

24. Zhao Z. Effects of Drinking Water Treatment Processes on Removal of Algal Matter and Subsequent Water Quality [dissertation]. Ontario: The University of Western Ontario; 2020.

25. Mohamed FM, El-Deen FN, Kamal AM. The Relationship between Algal Counting and Chemicals Consumption of Conventional Purification Systems at Qena Governorate, Egypt. Egypt J Aquat Res. 2020;24:161–172.
crossref pdf

26. Balcioglu IA, Alaton IA, Otker M, Bahar R, Bakar N, Ikiz M. Application of Advanced Oxidation Processes to Different Industrial Wastewaters. J Environ Sci Health. 2003;38:1587–1596.

27. Storhoff JJ, Lazarides AA, Mucic RC, Mirkin CA. What Controls the Optical Properties of DNA-Linked Gold Nanoparticle Assemblies? J Am Chem Soc. 2000;122:4640–4650.

28. Wang G-S, Hsieh S-T, Hong C-S. Destruction of humic acid in water by UV light-catalyzed oxidation with hydrogen peroxide. Water Res. 2000;34:3882–3887.

29. Lee TG, Bae HK. Growth characteristics of Microcystis aeruginosa and their effects on coagulation process efficiency. Wat Sci Tech: Wat Sup. 2012;12:124–131.
crossref pdf

30. Qi J, Lan H, Liu R, Liu H, Qu J. Efficient Microcystis aeruginosa removal by moderate photocatalysis-enhanced coagulation with magnetic Zn-doped Fe3O4 particles. Water Res. 2020;171:115448

Fig. 1
Turbidity changes for each cell state of Microcystis aeruginosa dependent on coagulant dose.
Fig. 2
Residual aluminum changes for each state of Microcystis aeruginosa dependent on coagulant dose.
Fig. 3
E254 changes for each cell state of Microcystis aeruginosa dependent on coagulant dose.
Fig. 4
E260 changes for each cell state of Microcystis aeruginosa dependent on coagulant dose.
Table 1
Chemical Composition of M11 Medium
M11 medium
Composition CaCl2·2H2O 0.004%
Fe-citrate 0.0006%
NaNO3 0.01%
K2HPO4 0.001%
MgSO4·7H2O 0.0075%
Na2CO3 0.002%
H2O 99.9749%
PDF Links  PDF Links
PubReader  PubReader
Full text via DOI  Full text via DOI
Download Citation  Download Citation
Editorial Office
464 Cheongpa-ro, #726, Jung-gu, Seoul 04510, Republic of Korea
TEL : +82-2-383-9697   FAX : +82-2-383-9654   E-mail : eer@kosenv.or.kr

Copyright© Korean Society of Environmental Engineers. All rights reserved.        Developed in M2community
About |  Browse Articles |  Current Issue |  For Authors and Reviewers